Fibrolase is a metalloproteinase isolated from the venom of the southern copperhead (Agkistrodon contortrix contortrix). It is typical of the small venom metalloproteinases of approximately 23 kDa. The enzyme possesses proteolytic activity directed to the cleavage of the .alpha.- and .beta.-chains of fibrin and fibrinogen. In that fibrin is a major component of occlusive thrombi, the degradative action of fibrolase leads to thrombus lysis and elimination. Fibrinolytic activity of fibrolase has been examined in both test tube and animal models. The enzyme has been shown to effectively lyse fibrin clots in-vitro [Guan, A. L., et al., Arch. Biochem. Biophys., 289:197-207 (1991)] and in-vivo [Markland, F. S., et al., Circulation, 90:2448-2456 (1994); Markland, F. S., in Natural Toxins II (Singh, B. R. and Tu, A. T., eds.), pp 427-438, Plenum Press, New York (1996)]. While fibrolase can degrade mature thrombi it has no effect on the formation of these structures.
Although fibrolase degrades fibrin(ogen) in the test tube, in the circulation the enzyme is efficiently inactivated by alpha-2 macroglobulin (.alpha.2M). To enable complete thrombus dissolution in vivo a modification to fibrolase must be made to block its rapid inactivation by .alpha.2M. .alpha.2M is a general protease inhibitor present in the circulatory system at fairly high concentrations (.congruent.3 .mu.M). This inhibitor has the ability to bind to and sequester small proteases and remove them from the circulation via the formation of a covalent bond between the proteinase and the very large, 720 kDa, tetrameric inhibitor molecule. Interactions between proteinases and .alpha.2M are sterically influenced and appear to be directly related to the size of the proteinase [Werb, Z., et al., Biochemical Journal, 139:359-368 (1974)]. A 68 kDa hemorrhagic metalloproteinase from Crotalus atrox is not inhibited by .alpha.2M, while another closely related but smaller, 23 kDa, metalloproteinase is rapidly and effectively bound and inhibited by .alpha.2M [Baramova, E. N., et al., Biochemistry, 29:1069-1074 (1990)]. Once bound to .alpha.2M the proteinase is essentially removed from circulation, unable to act on the target molecule, in the case of fibrolase, a thrombus.